Abstract
We describe here biochemical characterization of the 20 S proteasome from the parasitic protozoan Trypanosoma brucei. Similar to the mammalian proteasome, the T. brucei proteasome is made up of seven alpha- and seven beta-subunits. Of the seven beta-type subunits, five contain pro-sequences that are proteolytically removed during assembly, and three of them are predicted to be catalytic based on primary sequence. Affinity labeling studies revealed that, unlike the mammalian proteasome where three beta-subunits were labeled by the affinity reagents, only two beta-subunits of the T. brucei proteasome were labeled in the complex. These two subunits corresponded to beta2 and beta5 subunits responsible for the trypsin-like and chymotrypsin-like proteolytic activities, respectively. Screening of a library of 137,180 tetrapeptide fluorogenic substrates against the T. brucei 20 S proteasome confirmed the nominal beta1-subunit (caspase-like or PGPH) activity and identified an overall substrate preference for hydrophobic residues at the P1 to P4 positions in a substrate. This overall stringency is relaxed in the 11 S regulator (PA26)-20 S proteasome complex, which shows both appreciable activities for cleavage after acidic amino acids and a broadened activity for cleavage after basic amino acids. The 20 S proteasome from T. brucei also shows appreciable activity for cleavage after P1-Gln that is minimally observed in the human counterpart. These results demonstrate the importance of substrate sequence specificity of the T. brucei proteasome and highlight its biochemical divergence from the human enzyme.
Highlights
Proteasomes are multisubunit protein complexes responsible for the programmed degradation of protein substrates involved in a number of diverse cellular processes, including cell cycle progression, transcriptional regulation, and antigen presentation by major histocompatibility complex class I molecules [1, 2]
These N-terminal threonine residues are generated by proteolytic cleavage of extended precursor proteins during proteasome assembly, which serves as the primary mechanism to prevent pre-mature activation of incompletely assembled complexes [18]
Analysis of Biochemical Properties of the Subunits of T. brucei 20 S Proteasome—Samples of purified T. brucei 20 S proteasome were resolved by two-dimensional gel electrophoresis
Summary
T. brucei Cultures and 20 S Proteasome Purification—T. brucei 427 strain procyclic form cells were cultured in Cunningham’s medium supplemented with heat-inactivated fetal calf serum at a final concentration of 10% [32]. Subunit N-terminal Sequence Determination—Two-dimensional protein gel analysis of the purified T. brucei 20 S proteasome was carried out as previously described [31]. Purified T. brucei 20 S proteasome (24 nM for assays without PA26, 2.4 nM for assays with 640 nM PA26) in a buffer containing 60 mM Tris-HCl, pH 7.5, 10 mM KCl, and 5 mM MgCl2 was preincubated at 37 °C for 10 min to allow for association of the 20 S proteasome with PA26. The purified 20 S proteasomes from human and T. brucei at a concentration of 20 nM were preincubated for 10 min in the assay buffer containing, 60 mM Tris-HCl, pH 7.5, 10 mM KCl, and 5 mM MgCl2. Substrates were added at multiple concentrations, from 0.00001 to 1.75 mM (depending on solubility) to the preincubated enzyme, and substrate hydrolysis was monitored for 90 min at ex of 380 nm and em of 450 nm in a Molecular Devices Gemini XS microtiter plate reader. Data were fit using non-linear regression to the Michaelis-Menten equation and values for kcat, Km, and kcat/Km were determined
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